1. Field of the Invention
The present invention relates to an improved chemically modified hyaluronan preparation (hylan) characterized by novel chemical, physicochemical and rheological properties, and novel methods for obtaining such preparation.
2. The Prior Art
Hyaluronan, sometimes hereinafter also referred to as a hyaluronic acid (HA) is a naturally occuring high molecular weight glycosaminoglycan having a repeating disaccharide unit of D-glucuronic acid and N-acetylglucosamino-2-acetamido-2-desoxy-D-glucose joined by .beta.1.fwdarw.3 glucosidic bond. The disaccharides are joined to form an unbranched uncrosslinked polysaccharide chain by .beta.1.fwdarw.4 glucosidic bonds.
HA is found in animal tissues such as umbilical cord, vitreous, synovial fluid, rooster and chicken combs, skin, etc. The molecular weight of purified HA has been reported in the literature to be within the range of 50,000 to 8,000,000 depending on the source, method of isolation and method of determination of molecular weight (Balazs, E. A., Fed. Proceed. 17, 1086-1093 (1958).
Several methods have been suggested for recovery and purification of HA from animal tissues and bacterial cultures. Among these are enzymatic digestion of proteins (E. D. T. Atkins, C. F. Phelps and J. K. Sheehan, Biochem. J. 128, 1255-1263, (1972); R. Varma, R. S. Varma, W. S. Alten and A. H. Wardi, Carbohydr. Res. 32, 386-395, (1974); treatment with ion exchange resins (T. C. Laurent, J. Biol. Chem. 216, 253-271, (1955); E. R. Berman, Biochim. Biophys. Acta 58, 120-122 (1962); precipitation with cationic surfactants (T. C. Laurent, M. Ryan, and A. Pietruszkiewicz, Biochem. Biophys. Acta 42, 476-485 (1960)); treatment with trichloroacetic acid (H. Hofmans, O. Schmut, H. Sterk, and H. Koop, Naturforsch. 34c, 508-511 (1979); D. Schmut, and H. Hofmans, Biochim Biophys. Acta 673, 192-196 (1981)); preparative density gradient sedimentation (P. Silpanata, J. R. Dunstone, A. G. Ogston, Biochem. J. 109, 43-50 (1968)); and electrodeposition (S. Roseman, D. R. Watson, J. F. Duff, and W. D. Robinson, Annals Rheumatic Diseases, 15, 67-68 (1955)). One can also use a method which contains several different treatments, e.g., enzymatic digestion and precipitation with cetylpyridinium chloride (J. E. Scott, Biochem. J., 62, 31 (1956)).
The principal problem encountered in recovering HA from any biological source involves separating the polymer (HA) from proteins and other biological polymers which are extracted from the tissue along with the HA. Depending upon the raw material, the amount of undesirable polymers can be very large, exceeding by many times the amount of HA. The methods of HA recovery cited above are all used for the preparation of HA in laboratories but can hardly be used for large scale production of HA because of various drawbacks inherent in each of those methods.
The most advanced method for HA recovery and purification on an industrial scale is described in U.S. Pat. No. 4,141,973 (E. A. Balazs). According to this method, an ultrapure HA with a protein content of less than 0.5% by weight and a molecular weight more than 1,200,000 is obtained by water extraction from rooster combs or human umbilical cord. Proteins and other substances are removed with several chloroform extractions at varying pH values. In a chloroform extraction of the water extract an interface layer is formed in which denatured proteins and other substances are collected. Some substances, e.g., fats, are solubilized, probably in the chloroform phase. The process can also include a treatment with a proteolytic enzyme, e.g. pronase. By combining several quite elaborate treatments a process has been developed which allows one to obtain a pyrogen-free, non-inflammatory fraction of HA. This product is presently being marketed as a 1% solution under the trademark Healon.RTM. and is used in viscosurgery where it protects tissues against mechanical damage, provides space and permits manipulation of tissues during surgery (E. A. Balazs, Healon, J. Wiley and Son, N.Y., 1983, pp 5-28).